【作者】 冯永辉；

【导师】 吕昌龙；

【作者基本信息】 中国医科大学 ， 免疫学， 2010， 硕士

【摘要】 前言大蒜是一种含有多种有机硫化合物的植物,其中最重要的有效成分之一是大蒜素。人们用大蒜来治疗感染,枪伤,蛇咬,腹泻,风湿,心脏病,糖尿病以及其它的一些生理功能紊乱,人们在实验中还发现其具有降血脂、降血压、降血糖、抗肿瘤、抗菌、抗氧化和免疫调节等功效。大蒜来源的有机硫化合物对一些人类肿瘤,如前列腺癌,胸腺癌,结肠癌,肺癌,膀胱癌等有很好的抑制作用。大蒜的化学组成十分复杂,其中有生物活性的成分超过100种。其中最重要和最独特的是有机硫化合物的含量非常高,是其他含有高硫的蔬菜如洋葱、椰菜的4倍多。近年来日本学者发现在控制湿度和温度的情况下,普通的白大蒜可以经过处理转化为黑大蒜。黑大蒜去除了大蒜的异味,带有甜味,口感好,无需再经处理即可直接食用。已有研究报道黑大蒜较白大蒜含有更多的氨基酸、有机硫和S-烯丙基-L-半胱氨酸,因此与白大蒜相比,更具有应用前景。然而目前有关黑大蒜对机体免疫功能影响国内外尚未见报道。为此,本实验应用自制的黑大蒜提取物溶液,将腹腔注射给小鼠,观察了黑大蒜提取物作为免疫调节剂的作用,为黑大蒜的实际应用进一步提供理论和实验依据。实验材料一、主要试剂黑大蒜提取物(干粉,由日本弘前大学佐佐木甚一教授惠赠),RPMI1640培养基,小牛血清,0.01M PBS缓冲液,红细胞裂解液,LDH底物液,Griess试剂,IL-2、IFN-γ、IL-4、TNF-αELISA试剂盒。二、实验动物BALB/c小鼠,6-8周龄,SPF级,购自中科院上海实验动物中心。实验方法一、实验动物分组及处理40只BALB/c小鼠随即分为8组,其中第1-7组为实验组,第8组为对照组。将黑大蒜提取物用生理盐水配制成1mg/ml的溶液。实验组每天腹腔注射100μl的黑大蒜提取物溶液,对照组每天腹腔注射100μl的生理盐水。连续注射5天。于末次注射后的1-7天,从第1组开始,每天处死一组小鼠,第8组小鼠于第7天处死。二、样品的采集与检测1、脾细胞分离及其悬液的制备颈椎脱位处死小鼠,常规制备脾细胞悬液,调整细胞浓度至1×107／ml。2、ELISA法检测脾细胞培养上清中Th1细胞因子IL-2、TNF-α、IFN-γ以及Th2细胞因子IL-4的分泌水平3、LDH释放法检测NK细胞的杀伤活性4、Griess法检测脾细胞培养上清中NO分泌水平5、RT-PCR半定量技术检测脾细胞FasL mRNA的表达水平三、统计学处理实验数据经SPSS 16.0统计分析软件应用t检验进行统计处理,P<0.05有显著性差异。结果1、NK细胞杀伤活性的检测结果在连续5天给予BALB/c小鼠注射黑大蒜提取物后,比较实验组和对照组小鼠的NK细胞杀伤活性,结果表明：与对照组(5.82±1.38%)相比,黑大蒜提取物处理4-6天后,实验组小鼠的NK细胞杀伤活性显著性升高,并在第5天达到峰值(11.21±1.17%),随后下降。2、脾细胞培养上清中细胞因子水平的检测结果本实验中我们检测了Th1型细胞因子IL-2、IFN-γ和TNF-α和Th2细胞因子IL-4的分泌水平。我们发现在给予小鼠连续注射5天黑大蒜提取物后,IL-2的分泌水平在处理后第6天有明显升高,与对照组(20.14±3.88pg/ml)相比,有显著的统计学意义(P<0.05)。TNF-α的水平从处理后第1天就开始明显升高(P<0.01),在第3天达到峰值(68.73±0.64pg/ml),随后下降,但一直到第7天,均高于对照组(27.36±8.30pg/ml)。IFN-γ的分泌水平在处理结束后也有显著升高,其中在第3天达到峰值(105.05±55.73 pg/ml),随后下降,到第7天接近对照组水平(17.38±1.19 pg/ml)。Th2细胞因子IL-4的水平在处理后其分泌水平一直低于对照组(19.98±2.13 pg/ml),其中第3、4、5天的表达水平均显著低于对照组(P<0.01),但是变化均在基线范围内。3、脾细胞培养上清中NO含量的检测结果从黑大蒜提取物处理后的第3天到第7天,实验组小鼠NO2-始终处于较高的水平,与对照组相比(11.58±0.86μM),均具有显著性差异(P<0.001)。4、脾细胞FasL mRNA表达水平的检测结果通过半定量RT-PCR技术检测了小鼠脾细胞FasL mRNA的表达水平。处理结束后的第1天至第7天,各天脾细胞FasL mRNA的表达水平与对照组相比均未见显著性差异(P>0.05)。结论1、黑大蒜提取物可以增强NK细胞的杀伤活性。2、FasL mRNA的表达水平并未增高,提示黑大蒜提取物未诱导杀伤细胞通过Fas/FasL途径发挥效应。3、黑大蒜提取物可使脾细胞NO的分泌水平明显增高,提示黑大蒜提取物可能有增强固有免疫细胞巨噬细胞活性的作用。4、黑大蒜提取物能增强IL-2、IFN-γ、TNF-α的产生水平,提示黑大蒜提取物能够促进Th1型免疫应答。更多还原

【Abstract】 IntroductionGarlic is a kind of plant containing many organic sulfur compounds, and one of the most effective components is allicin. People used garlic to treat infection, wound, snake bite, diarrhea, rheumatism, heart diseases, diabetes, and other physiologic function disorders. Researches on the garlic reveal lowering the blood fat, blood pressure and blood sugar, anti-cancer, antibiosis and immunomodulator. The organic sulfur compounds derived from garlic can inhibit some cancers, such as prostate cancer, breast cancer, colon cancer, lung cancer, and bladder cancer. The chemical constituents of garlic are very complex containing over 100 different compounds which are regarded as contributing to beneficial biological effects. The most important and unique feature is the high content of organo-sulfur substance, which are at least four times more than other high sulfur-containing vegetables such as onion, broccoli, and cauliflower. Recent years, some Japanese researchers have developed a new type of black garlic by processing ordinary garlic under control of the temperature and humidity. The irritating odor is removal and Black garlic with fruity taste is readily edible without further treatments. There has been report on the black garlic containing more amino acid, organic sulfur and S-allyl-L-cysteine than ordinary garlic. However, there are no reports home and abroad on the black garlic affecting the immune functions. Therefore, we intraperitoneally injected the mice with black garlic extracts solution and observe the effects of black garlic extracts as a type of immunomodulator, and provide the immunological and experiment evidences for the practical application.To further approach the black garlic on the immune function in mcie, we give the normal BALB/c mice intraperitoneal injection with BGE solution, and detect the effects on the immune function. Materials1. RegentsBGE (black garlic extracts, dry powder, granted by Prof. Sasaki from Hirosaki University), RPMI 1640 Medium, fetal bovine serum,0.01 M phosphate buffer solution, Red blood cells lysis solution, LDH substrate solution, Griess reagent, ELISA Kit for IL-2, IFN-γ, TNF-a, and IL-4.2. AnimalFemale BALB/c mice,6-8 weeks old (Academia Sinica Shanghai experimental animal center).Methods1. Animal grouping and treatments40 female BALB/c mice were randomly divided into eight groups, and Group 1-7 were test groups and Group 8 was control group.The black garlic extract solution was prepared with normal saline and the concentration was 1 mg/ml. Mice in test groups were intraperitoneally injected 0.1 ml black garlic extract solution and that in control group with normal saline for successive five days. From the first day to the seventh day after the last injection, one test group of mice were sacrificed each day and mice in the control group were executed on the seventh day.2. Samples collection and analysis(1) Separate spleen cells and prepare splenic suspensionSacrifice the mice by cervical location, prepare the spleen cell suspension by conventional method, and adjust the cell concentration into 1×107/ml.(2) Th1 cytokines IL-2, TNF-a,IFN-γand Th2 cytokine IL-4 in spleen cell culture supernatant were detected by Elisa.(3) The cytotoxicity of NK cells was detected by LDH release assay(4) The NO secreting level of cultured spleen cells by Griess method(5) FasL mRNA level of spleen cells was detected by RT-PCR semi-quantity assay(6) Statistical analysisT-test (SPSS 16.0 software) was applied to compare the experimental data between test group and control group. Results1. The result of cytotoxicity of NK cellsFollowing by giving the black garlic solution by intraperitoneally injection for five successive days, the data of the cytotoxicity of NK cells in test group suggests that the toxicity of NK cells was significantly elevated from the fourth to sixth days post treatment compared with control group (5.82±1.38%) and reached the peak value (11.21±1.17%), then decreased.2. The detection results of cytokines level in cultured spleen cells supernatantWe detected the secreting level of Thl cytokines IL-2, IFN-y, TNF-a and Th2 cytokine IL-4 after treat the mice with the BGE solution and the results indicate that the IL-2 secreting level was significantly elevated on the sixth day post treatment, there existing statistical significance (P< 0.05) compared with control group (20.14±3.88 pg/ml). TNF-a secreting level was significantly increased (P＜0.01) from the first day after treatment, and reached the peak value (68.73±0.64 pg/ml)on the third day, then decreased. However, it’s still higher than that in control group (27.36±8.30 pg/ml) on the seventh day. IFN-y secreting level was also increased after treatment, and reached the peak value (105.05±55.73 pg/ml), then decreased to the level slose to the control group (17.38±1.19 pg/ml) on the seventh day. Th2 cytokine IL-4 secreting level has been lower than control, and the expressing level was significantly lower (P< 0.01) than that in control group (19.98±2.13 pg/ml), but the variation was also in the base line.3. The detection results of NO level in cultured spleen cell supernatantNO2- level was always higher than control group (11.58±0.86μM) from the third to seventh day after treatment with BGE, there existing statistical significance (P< 0.01).4. The detection results of spleen cells’FasL mRNA levelWe detected the spleen cells FasL mRNA expressing level by Semi-quantity RT-PCR. The results suggested there was no difference of FasL mRNA expressing level between test and control group. Conclusion1. BGE can increase the cytotoxicity of NK cells.2. FasL mRNA expressing level is not elevated which suggests that BGE doesn’t induce killer cells’cytotoxicity by Fas/FasL pathway.3. Black garlic can highly increase the NO secreting level of cultured spleen cell supernatant, which suggests that BGE can enhance macrophage’s activity.4. BGE can enhance the production of IL-2, IFN-γ,TNF-αin the supernatant of the cultured spleen cells, which suggests that BGE can enhance Thl immune response.更多还原